1. Field of the Invention
The present invention relates to an enhanced luminescent or luminometric assay, particularly immunoassay, and to a diagnostic kit for use in the assay. The luminescent and luminometric assays with which the present invention is concerned are those depending on a chemiluminescent reaction (a chemical reaction that results in the emission of light). The luminescent emission is generally of sufficient duration to enable the light emitted to be detected or measured, and thereby to allow the detection or quantification of an analyte. The chemiluminescent reaction with which this invention is concerned is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol or isoluminol, with an oxidant, especially hydrogen peroxide, and a peroxidase enzyme which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by emission of light.
2. Description of the Prior Art
Luminescent and luminometric assays making use of the above-mentioned peroxidase-catalysed oxidation of a DPD include three major types.
a. Assays wherein a chemiluminescent compound is used directly to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. The chemiluminescent DPD such as luminol or isoluminol is normally conjugated to a ligand. Chemiluminescence can be detected by adding peroxidase and an oxidant to the reacted conjugate. See for example UK Patent Specifications 2026690A (Miles Laboratories, Inc.), especially page 7 and 2008247A (The Welsh National School of Medicine), especially page 8, Example 7.
b. Assays wherein catalysts or cofactors of luminescent reactions are used as labels, and the luminescent reaction is used to detect or quantitate the label. Normally an enzyme, frequently horseradish peroxidase is conjugated to a ligand. This category therefore includes peroxidase ELISAs.
c. Assays wherein luminescent reactions are used to determine the products formed by the action of enzyme labels other than peroxidase on suitable substrates. An example of this type of assay is the determination of antibody-linked glucose oxidase by reacting the enzyme/antibody reagent with glucose to form hydrogen peroxide and then measuring the amount of hydrogen peroxide produced by adding luminol and a peroxidase catalyst under controlled conditions to initiate a luminescent reaction.
Examples of assays which are not immunoassays but which make use of a luminescent reaction include:
a. An elastase assay based on the release of peroxidase from an insoluble peroxidase-elastin preparation,
b. A glucose assay based on co-immobilised glucose oxidase and peroxidase, and
c. An assay of a peroxidase enzyme, a 2,3-dihydro-1,4-phthalazinedione, or an oxidant, such as hydrogen peroxide, when these materials are neither labels nor the products of labels.
A review of luminescent and luminometric assays is given by T P Whitehead et al., Clinical Chemistry 25 1531-1546 (1979).
The sensitivity of immunoassays is determined in part by the lower limit for detection of the label or the product of the label. In the case of luminescent or luminometric immunoassays their sensitivity will depend partially on the light emitted in the luminescent reaction per unit of labelled material.
Peroxidases catalyse the oxidation of a wide variety of organic compounds. Advantage has been taken of these reactions for assay purposes by measuring colour formed, fluorescence (=photoluminescence, caused by the action of light on the reaction product) or chemiluminescence (caused by release of light by the reaction carried out in the dark).
Many different colour-forming substrates are mentioned in a review of prior art in Research Disclosure No 16034, anonymous, 19-24 (August 1977). They include aromatic monoamines, eg aniline and its derivatives, diamines such as o- and p-phenylenediamine and benzidine, monophenols, polyphenols, aromatic acids, leuco dyes, coloured dyes such as 2,6-dichlorophenolindophenol, various biological substances, gums, various iodides, bilirubin and the dyes 2,2'-azino-di-(3-ethylbenzthiazoline)-6-sulphonate (ABTS) and 3,3'-diaminobenzidine. The Research Disclosure paper also describes other colour-formers, viz. (1) a sulphonyl hydrazone containing an N-heterocyclic ring in combination with a coupler, eg a phenol or aromatic amine, and (2) a triarylimidazole. Other colour-forming substrates have been summarised by D J Capaldi et al., Analytical Biochemistry 129 329-336 (1983). These authors refer to the redox substrates ABTS and o-dianisidine (3,3'-dimethoxybenzidine) and to colour-forming combinations of compounds including 4-aminoantipyrine with a phenol, 3-methyl-2-benzothiazolinone hydrazone (MBTH) together with N,N-dimethylaniline, 2-hydroxy-3,5-dichlorobenzene sulphonate or 3-(N,N-dimethylamino)benzoic acid.
The use of 4-aminoantipyrine with a phenol coupler is described by C. C. Allain et al., Clinical Chemistry 20, 470 to 475 (1974) and with N,N-dimethylaniline as a coupler by T Kikuchi et al., Chemical Abstracts 94, 1799v (1981) and Y Tsutomu, Chemical Abstracts 94 79759b (1981). See also M Tsuda, Chemical Abstracts 93, 65423e (1980) and Eikenchemical Ltd, 94 135589k (1981). P Josephy et al., J Biol. Chem. 257, 3669-3675 (1982) describes a study of the HRP-catalysed oxidation of the colour-forming compound 3,5,3',5'-tetramethylbenzidine.
Compounds which on oxidation by H.sub.2 O.sub.2 and peroxidase give fluorescent products include tyramine (4-hydroxyphenethylamine) and homovanillic acid: K Matsuoka et al., Chem. Pharm. Bull. 27, 2345-2350 (1979), 4-hydroxyphenylacetic acid: S D Lidofsky et al., Proc. Nat. Acad. Sci. USA 78, 1901-1905 (1980), various phenols: K Zaitsu et al., Analytical Biochemistry 109, 109-113 (1980) and the amino acid tyrosine: J Williams et al., Biochem. J. 121, 203-209 (1971).
Luminescent substrates other than DPDs include polyphenols such as pyrogallol, lophine which is 2,4,5-triphenylimidazole, acridinium compounds and diaryl oxalates, see the review by T P Whitehead et al. supra.
Enhancement of peroxidase-catalysed oxidations of various organic compounds by various enhancers (which increase reaction rate or intensity of the colour or luminescence measured) has been reported as shown in the following Tables 1 and 2.
TABLE 1 __________________________________________________________________________ Enhancement of non-luminescent reactions involving a peroxidase Reactants Enhancer Reference __________________________________________________________________________ o-Methoxyphenol/H.sub.2 O.sub.2 /HRP KCl, NaCl, LiCl or A (Guaiacol) sodium sulphate 3,3'-Dimethoxybenzidine/H.sub.2 O.sub.2 /HRP Ammonia and ammonium B p-Phenylenediamine/H.sub.2 O.sub.2 /HRP salts, pyridine and imidazole Diaminobenzidine/H.sub.2 O.sub.2 /HRP Imidazole C ABTS and other colour-forming Various phenols D and fluorescent substrates Ascorbic acid/H.sub.2 O.sub.2 /HRP o-Methoxy phenol, E " p-Hydroxydiphenyl or " p-Methoxy phenol p-Cresol F " 1,3-Dihydroxybenzene G Thyroxine and H Thyroxine analogues Epinephrine/H.sub.2 O.sub.2 /HRP Thyroxine and H thyroxine analogues Ferrocyanide/H.sub.2 O.sub.2 /HRP Benzohydroxamic acid J 1-Anilino-8-naphthalene sulpho- Benzohydroxamic acid K nate/H.sub.2 O.sub.2 /HRP 2-p-Toluidinyl naphthylene-6- sulphonate/H.sub.2 O.sub.2 /HRP Indole-3-acetic acid/H.sub.2 O.sub.2 /HRP 2,4-Dichlorophenol L __________________________________________________________________________
TABLE 2 ______________________________________ Enhancement of luminescent reactions involving a peroxidase Reactants Enhancer Reference ______________________________________ Purpurogallin/H.sub.2 O.sub.2 Para-phenylenediamine M Pyrogallol/H.sub.2 O.sub.2 Ortho-phenylenediamine N ______________________________________